Abstract Results from the HIVE Center and from others have transformed the way that we think about the mechanistic basis of HIV DNA integration. Until quite recently, the consensus view was that an integrase tetramer, working in the context of the intasome nucleoprotein complex, catalyzed retroviral integration into chromatin. However, over the past year, the tetramer-centric view of retroviral integration has been exposed as overly simplistic. Work in part funded by this grant revealed that beta-retroviral integration is promoted by an integrase octamer. More recently, cryo-electron microscopy revealed that the structure of the HIV-1 strand transfer complex, the final intasome complex in the integration pathway, is polymorphic, containing both simple tetramer arrangements as well as higher-order dodecamers/hexadecamers. Higher-order complex formation moreover depended on the presence of the integrase-binding domain of the common integration co-factor LEDGF. These observations lead to several new questions in the field, which will be addressed in this grant application. For example, do intasome complexes that precede the strand transfer complex also comprise a mixture of different multimers, or, by its nature, does integration pathway maturation necessitate higher-order multimer formation? Several cutting edge approaches, including single-particle cryo-electron microscopy and single-molecule fluorescence imaging, will be used to characterize the mechanistic basis of intasome assembly and function as the complexes mature along the HIV-1 integration pathway. In addition to assessing the role of LEDGF in pathway maturation, the LEDGF structure will be determined bound to nucleosomes, and as the tether that links the intasome to the nucleosome. In addition to directing integration into active genes, LEDGF has recently been implicated in the regulation of HIV latency. Although the capsid binding CPSF6 factor plays a greater role than LEDGF to direct integration to active chromatin, a potential role for CPSF6 in regulating HIV latency is unknown. Here, we will comprehensively address the roles of the two main integration targeting cofactors, LEDGF and CPSF6, in the establishment and regulation of HIV-1 latency in cell line models as well as in primary T cells. The completion of this project will provide for the structural basis of HIV-1 integration into chromatin and the consequences of disrupting these pathways on the establishment and regulation of HIV proviral latency.